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通过PCR方法筛选160株苏云金芽孢杆菌,其中94株含有zmaR基因。测定了含zmaR基因的菌株培养物上清液对草生欧文氏杆菌(Erwiniaherbicola)的抑菌活性,结果表明有67株菌有抑菌活性,其中21株抑菌活性较高。经鉴定,抑菌活性较高的G03菌株含有cry1Ac、cry1Aa、cry1Ca和cry2Ab等高毒力杀虫基因,并从G03中克隆了zmaR全长基因,完成了序列测定。该基因编码区为1125bp,由核苷酸序列推导的氨基酸残基组成为375个,分子量为43.5kD,等电点pI4.945。通过载体pET-21b将zmaR基因导入大肠杆菌BL21,可正常表达43.5kD蛋白,并使宿主菌产生对ZwittermicinA的抗性。
160 strains of Bacillus thuringiensis were screened by PCR, of which 94 contained zmaR gene. The antibacterial activity of the strain culture supernatant containing zmaR gene against Erwinia herbbicola was determined. The results showed that 67 strains had antibacterial activity, of which 21 strains had high antibacterial activity. The strain G03 with high antibacterial activity was identified as a highly toxic insecticidal gene of cry1Ac, cry1Aa, cry1Ca and cry2Ab. The full-length zmaR gene was cloned from G03, and the sequence determination was completed. The coding region of this gene was 1125bp. The amino acid residues deduced from the nucleotide sequence constituted 375 amino acids with a molecular mass of 43.5kD and an isoelectric point of pI4.945. Introduction of the zmaR gene into E. coli BL21 by the vector pET-21b normally expressed 43.5 kD of protein and allowed the host bacteria to develop resistance to ZwittermicinA.